Strategies for Measuring Damage and Repair in Gene-Sized Specific DNA Sequences

  • Charles A. Smith
  • Philip C. Hanawalt


Interest in how the efficiency of DNA repair might vary among specific categories of cellular DNA dates almost to the origin of the “repair replication” technique, which quantifies the short stretches of DNA synthesized during excision repair (Pettijohn and Hanawalt, 1964). It has always been clear that the biological consequences of DNA damage to the cell or organism would depend strongly on the functional role of the particular segment of DNA suffering the damage. Early studies were confined to comparing repair in classes of DNA that were in relative abundance and could be physically isolated for analysis such as chloroplast and mitochondrial DNA, and genomic satellite DNA. Later, the repair of the highly repetitive alpha DNA sequences in African green monkey cells was investigated in detail using a variety of techniques. This was made possible by the abundance of this alpha DNA species; 8% of the DNA can be easily isolated as pure 172-base-pair fragments by digestion by HindIII and gel electrophoresis (Zolan et al., 1982). These investigations (reviewed in Smith, 1987) demonstrated complex differences in the repair of this nontranscribed sequence as compared to the remaining, bulk DNA, and gave impetus to efforts to develop methods for studying repair in active genes.


Repair Patch Cyclobutane Pyrimidine Dimer DHFR Gene Lesion Frequency Adenine Phosphoribosyltransferase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Baird, W. M., Smith, C. A., Spivak, G., Mauthe, R. J., and Hanawalt, P. C. (1994). Analysis of the fine structure of the repair of anti-benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts in mammalian cells by laser-induced strand cleavage. Polycyclic Aromatic Compounds 6:169–176.CrossRefGoogle Scholar
  2. Bohr, V. A., and Okumoto, D. S. (1988). Analysis of pyrimidine dimer repair in defined genes, in:DNA Repair: A Laboratory Manual of Research Procedures, Volume III (E. C. Friedberg and P. C. Hanawalt, eds.), Dekker, New York, pp. 347–366.Google Scholar
  3. Bohr, V. A., Smith, C. A., Okumoto, D. S., and Hanawalt, P. C. (1985). DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40:359–369.PubMedCrossRefGoogle Scholar
  4. Chen, R. H., Maher, V. M., Brouwer, J., van de Putte, P., and McCormick, J. J. (1992). Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts. Proc. Natl Acad. Sci. USA 89:5413–5417.PubMedCrossRefGoogle Scholar
  5. Denissenko, M. F., Venkatachalam, S., Yamasaki, E. F., and Wani, A. A. (1994). Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR. Nucleic Acids Res. 22:2351–2359.PubMedCrossRefGoogle Scholar
  6. Islas, A. L., Vos, J.-M., and Hanawalt, P. C. (1991). Differential introduction and repair of psoralen-DNA interstrand crosslinking in specific human genes. Cancer Res. 51:2867–2873.PubMedGoogle Scholar
  7. Leadon, S. A. (1988). Immunological probes for lesions and repair patches in DNA, in:DNA Repair: A Laboratory Manual of Research Procedures, Volume III (E. C. Friedberg and P. C. Hanawalt, eds.), Dekker, New York, pp. 311–326.Google Scholar
  8. Leadon, S.A., and Cooper, P. K. (1993). Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome. Proc. Natl. Acad. Sci. USA 90:10499–10503.PubMedCrossRefGoogle Scholar
  9. Leadon, S. A., and Lawrence, D. A. (1992). Strand-selective repair of DNA damage in the yeast GAL7 gene requires RNA polymerase II. J. Biol. Chem. 267:23175–23182.PubMedGoogle Scholar
  10. Lommel, L., and Hanawalt, P.C. (1991). The genetic defect in the Chinese hamster ovary cell mutant UV61 permits moderate selective repair of cyclobutane pyrimidine dimers in an expressed gene. Mutat. Res. 255:183–191.PubMedCrossRefGoogle Scholar
  11. Madhani, H. D., Bohr, V. A., and Hanawalt, P.C. (1986). Differential DNA repair in a transcriptionally active and inactive proto-oncogene:c-abl and c-mos. Cell 45:417–423.PubMedCrossRefGoogle Scholar
  12. Mellon, I., Spivak, G., and Hanawalt, P. C. (1987). Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51:241–249.PubMedCrossRefGoogle Scholar
  13. Pettijohn, D., and Hanawalt, P. C. (1964). Evidence for repair-replication of ultraviolet damaged DNA in bacteria. J. Mol. Biol. 9:395–410.PubMedCrossRefGoogle Scholar
  14. Ruven, H. J., Seelen, C. M., Lohman, P. H., Mullenders, L. H., and van Zeeland, A. A. (1994). Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR, application of the method for analysis of strand-specific DNA repair. Mutat. Res. 315:189–195.PubMedCrossRefGoogle Scholar
  15. Scicchitano, D., and Hanawalt, P. C. (1989). Repair of N-methylpurines in specific DNA sequences in Chinese hamster ovary cells: Absence of strand specificity in the dihydrofolate reductase gene. Proc. Natl. Acad. Sci. USA 86:3050–3054.PubMedCrossRefGoogle Scholar
  16. Smith, C. A. (1987). DNA repair in specific sequences in mammalian cells. J. Cell Sci. Suppl. 6:225–241.CrossRefGoogle Scholar
  17. Smith, C. A. (1988). Repair of DNA containing furocoumarin adducts, in:Psoralen DNA Photobiology, Volume II (F. Gasparro, ed.), CRC Press, Boca Raton, FL, pp. 87–116.Google Scholar
  18. Spivak, G., and Hanawalt, P. C. (1992). Translesion DNA synthesis in the DHFR domain of UV-irradiated CHO cells. Biochememistry 31:6794–6800.CrossRefGoogle Scholar
  19. Spivak, G., and Hanawalt, P. C. (1995). Determination of damage and repair in specific DNA sequences, in:Methods: A Companion to Methods in Enzymology, Vol. 7, Academic Press, London, pp. 147–161.Google Scholar
  20. Tang, M. S., Pao, A., and Zhang, X. S. (1994a). Repair of benzo(a)pyrene diol epoxide-and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells. J. Biol. Chem. 269:12749–12754.PubMedGoogle Scholar
  21. Tang, M. S., Qian, M., and Pao, A. (1994b). Formation and repair of antitumor antibiotic CC-1065-induced DNA adducts in the adenine phosphoribosyltransferase and amplified dihydrofolate reductase genes of Chinese hamster ovary cells. Biochemistry 33:2726–2732.PubMedCrossRefGoogle Scholar
  22. Thomale, J., Hochleitner, K., and Rajewsky, M. F. (1994). Differential formation and repair of the mutagenic DNA alkylation product O6-ethylguanine in transcribed and nontranscribed genes of the rat. J. Biol. Chem. 269:1681–1686.PubMedGoogle Scholar
  23. Thomas, D. C., Morton, A. G., Bohr, V. A., and Sancar, A. (1988). General method for quantifying base adducts in specific mammalian genes. Proc. Natl. Acad. Sci. USA 85:3723–3727.PubMedCrossRefGoogle Scholar
  24. van Hoffen, A., Venema, J., Meschini, R., van Zeeland, A. A., and Mullenders, L. H. (1995). Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6–4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts. EMBO J. 14:360–367.PubMedGoogle Scholar
  25. Venema, J., van Hoffen, A., Karcagi, V, Natarajan, A. T., van Zeeland, A. A., and Mullenders, L. H. (1991). Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes. Mol. Cell. Biol. 11:4128–4134.PubMedGoogle Scholar
  26. Vos, J.-M. (1988). Analysis of psoralen monoadducts and interstrand crosslinks in defined genomic sequences, in:DNA Repair: A Laboratory Manual of Research Procedures, Volume III (E. C. Friedberg and P. C. Hanawalt, eds.), Dekker, New York, pp. 367–398.Google Scholar
  27. Wang, W., Sitaram, A., and Scicchitano, D. A. (1995). 3-Methyladenine and 7-methylguanine exhibit no preferential removal from the transcribed strand of the dihydrofolate reductase gene in Chinese hamster ovary B11 cells. Biochemistry 34:1798–1804.PubMedCrossRefGoogle Scholar
  28. Zolan, M. E., Cortopassi, G. A., Smith, C. A., and Hanawalt, P. C. (1982). Deficient repair of chemical adducts in alpha DNA of monkey cells. Cell 28:613–619.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Charles A. Smith
    • 1
  • Philip C. Hanawalt
    • 1
  1. 1.Department of Biological SciencesStanford UniversityStanfordUSA

Personalised recommendations